Planktonic-cell yield of a pseudomonad biofilm

Biofilm cells differ phenotypically from their free-floating counterparts. Differential growth rates in biofilms are often referred to, particularly in response to limited diffusion of oxygen and nutrients. We observed growth rates of attached Pseudomonas sp. strain CT07 cells that were notably higher than the maximum specific growth rate measured in batch culture. Despite dilution rates in continuous flow cells that exceeded the maximum planktonic specific growth rate by 58 times, sampling of the effluent revealed >10(9) cells ml(-1), suggesting that biofilms function as a source of planktonic cells through high cell yield and detachment. Further investigation demonstrated considerable planktonic cell yield from biofilms as young as 6 h, indicating that detachment is not limited to established biofilms. These biofilm-detached cells were more sensitive to a commercial biocide than associated biofilm- and chemostat-cultivated populations, implying that detached biofilm cells exhibit a character that is distinct from that of attached and planktonic cell populations.     

COCCOLITH PRODUCTION AND DETACHMENT BY EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE)1

Laboratory experiments were performed with the prymnesiophyte Emiliania huxleyi (Lohm.) Hay and Mohler, strain 88E, to quantify calcification per cell, coccolith detachment, and effects of coccolith production on optical scattering of individual cells. ¹⁴C incorporation into attached and detached coccoliths was measured using a bulk filtration technique. ¹⁴C-labeled cells also were sorted using a flow cytometer and analyzed for carbon incorporation into attached coccoliths. The difference between the bulk and flow cytometer analyses provided a ¹⁴C-based estimate of the rate of production of detached coccoliths. Coccolith production and detachment were separated in time in batch cultures, with most detachment happening well after calcification had stopped. Accumulation of coccoliths was maximum at the end of logarithmic growth with 50–80 coccoliths per cell (three to five complete layers of coccoliths around the cells). Net accretion rates of coccoliths were on the order of 7 coccoliths· cell^?1·d^?1 while net detachment rates were as high as 15 coccoliths· cell^?1·d^?1 for stationary phase cells. Equal numbers of coccoliths were attached and detached early in logarithmic growth, and as cells aged, the numbers of detached coccoliths exceeded the attached ones by a factor of 6. Our results demonstrate pronounced charges of forward angle light scatter and 90° light scatter of cells as they grow logarithmically and enter stationary phase. Counts of loose coccoliths in batch cultures are consistent with the detachment of coccoliths in layers rather than individual coccoliths.     

Planktonic-Cell Yield of a Pseudomonad Biofilm

Biofilm cells differ phenotypically from their free-floating counterparts. Differential growth rates in biofilms are often referred to, particularly in response to limited diffusion of oxygen and nutrients. We observed growth rates of attached Pseudomonas sp. strain CT07 cells that were notably higher than the maximum specific growth rate measured in batch culture. Despite dilution rates in continuous flow cells that exceeded the maximum planktonic specific growth rate by 58 times, sampling of the effluent revealed >10⁹ cells ml⁻¹, suggesting that biofilms function as a source of planktonic cells through high cell yield and detachment. Further investigation demonstrated considerable planktonic cell yield from biofilms as young as 6 h, indicating that detachment is not limited to established biofilms. These biofilm-detached cells were more sensitive to a commercial biocide than associated biofilm- and chemostat-cultivated populations, implying that detached biofilm cells exhibit a character that is distinct from that of attached and planktonic cell populations.     

Modeling 1,2-dichloroethane biodegradation by <Emphasis Type="Italic">Klebsiella oxytoca va 8391</Emphasis> immobilized on granulated activated carbon

A mathematical model of the biodegradation of xenobiotics by microbial cells attached to particles of granulated activated carbon was developed. The model allowed the quantitative evaluation of different characteristics of the biofilm behavior: retarded microbial growth, increased concentration of immobilized cells compared to suspended cultures, potential cell detachment from the solid support and consequent independent growth of free cells. The applicability of the model was demonstrated for our own experimental data for 1,2- dichloroethane (DCA) biodegradation by Klebsiella oxytoca VA 8391 cells attached to granulated activated carbon. Two types of reactors, recirculated batch and continuous flow bioreactor, were studied. It was shown that in all investigated cases, the major contribution to DCA biodegradation was provided by the immobilized cells. Furthermore, immobilized cells were found to tolerate much higher substrate concentration and dilution rates in continuous culture than the free cells.     

The kinetics of affinity-mediated cell-surface attachment

Data and a semi-empirical model are presented that describe the affinity interaction of yeast cells with a Concanavalin A derivatised surface. The model uses 3 parameters to describe the time course of cell attachment from a flowing suspension of yeast cells, over a range of flow rates, and gives an effective global fit to the data obtained. Further modifications allow the effects of a soluble competitor (glucose) on binding to be quantified in terms of a saturation effect, and an effective global fit is obtained. A comparison was made between the relationship between steady-state attached fraction and applied shear with similar data reported earlier (Ming, F. et al, 1998) for the detachment of pre-adsorbed cells. This shows that there is an order of magnitude difference between the forces required to effect complete detachment in the two systems, and that the nature of the relationship between shear and attached fraction is profoundly different. The magnitude of this time-dependent stabilization might be explained in terms of a progressive reorientation of cell relative to the surface such that the number of bonds is maximized.     

Influence of flow rate and scaffold pore size on cell behavior during mechanical stimulation in a flow perfusion bioreactor

Mechanically stimulating cell-seeded scaffolds by flow-perfusion is one approach utilized for developing clinically applicable bone graft substitutes. A key challenge is determining the magnitude of stimuli to apply that enhances cell differentiation but minimizes cell detachment from the scaffold. In this study, we employed a combined computational modeling and experimental approach to examine how the scaffold mean pore size influences cell attachment morphology and subsequently impacts upon cell deformation and detachment when subjected to fluid-flow. Cell detachment from osteoblast-seeded collagen-GAG scaffolds was evaluated experimentally across a range of scaffold pore sizes subjected to different flow rates and exposure times in a perfusion bioreactor. Cell detachment was found to be proportional to flow rate and inversely proportional to pore size. Using this data, a theoretical model was derived that accurately predicted cell detachment as a function of mean shear stress, mean pore size, and time. Computational modeling of cell deformation in response to fluid flow showed the percentage of cells exceeding a critical threshold of deformation correlated with cell detachment experimentally and the majority of these cells were of a bridging morphology (cells stretched across pores). These findings will help researchers optimize the mean pore size of scaffolds and perfusion bioreactor operating conditions to manage cell detachment when mechanically simulating cells via flow perfusion.     

Bacterial adhesion to hydroxyl- and methyl-terminated alkanethiol self-assembled monolayers.

The attachment of bacteria to solid surfaces is influenced by substratum chemistry, but to determine the mechanistic basis of this relationship, homogeneous, well-defined substrata are required. Self-assembled monolayers (SAMs) were constructed from alkanethiols to produce a range of substrata with different exposed functional groups, i.e., methyl and hydroxyl groups and a series of mixtures of the two. Percentages of hydroxyl groups in the SAMs and substratum wettability were measured by X-ray photoelectron spectroscopy and contact angles of water and hexadecane, respectively. SAMs exhibited various substratum compositions and wettabilities, ranging from hydrophilic, hydroxyl-terminated monolayers to hydrophobic, methyl-terminated monolayers. The kinetics of attachment of an estuarine bacterium to these surfaces in a laminar flow chamber were measured over periods of 120 min. The initial rate of net adhesion, the number of cells attached after 120 min, the percentage of attached cells that adsorbed or desorbed between successive measurements, and the residence times of attached cells were quantified by phase-contrast microscopy and digital image processing. The greatest numbers of attached cells occurred on hydrophobic surfaces, because (i) the initial rates of adhesion and the mean numbers of cells that attached after 120 min increased with the methyl content of the SAM and the contact angle of water and (ii) the percentage of cells that desorbed between successive measurements (ca. 2 min) decreased with increasing substratum hydrophobicity. With all surfaces, 60 to 80% of the cells that desorbed during the 120-min exposure period had residence times of less than 10 min, suggesting that establishment of firm adhesion occurred quickly on all of the test surfaces.     

Effect of cadmium and zinc on attachment and detachment interactions of Pseudomonas fluorescens H2 with glass.

The physiological and physicochemical bases for the effect of 5, 10, 50, or 100 micrograms of Cd and Zn ml-1 on the attachment and detachment interactions of Pseudomonas fluorescens H2 with glass substrata were determined. Attachment and detachment varied with the type and concentration of metal and the time at which cells were exposed to the metal. The largely inhibitory effect of the metals on bacterial motility and physiological activity did not directly influence attachment. The amount of Cd or Zn accumulated by the cells increased with metal concentration and was greater for free than for attached cells. The hydrophobicity and negative and positive charges of the bacterial surfaces (measured by hydrophobic and electrostatic interaction chromatography) were increased by cell exposure to the metals, particularly after Cd treatment. Cells exposed to Cd prior to attachment showed increased adhesion. Zinc-treated cells did not. There was a positive correlation between adhesion and Cd concentration in the attachment solution. No such relationship existed for Zn. P. fluorescens H2 exposed to Cd prior to attachment desorbed similarly to untreated controls. Zinc pretreatment resulted in decreased desorption. Cells attached in 5 or 10 micrograms of Cd or Zn ml-1 detached less than those attached in 50 or 100 micrograms of Cd or Zn ml-1. The presence of Cd or Zn during detachment had little effect on desorption. The dominant influence of Cd and Zn on attachment and detachment appears to be through modification of the bacterial surface. In natural ecosystems, heavy metals may influence the distribution of bacteria between the solid and liquid phases.     

Tracking of injected and resident (previously injected) bacterial cells in groundwater using ferrographic capture

A high-resolution bacterial tracking technique, ferrographic capture, was used to enumerate fluorescent-stained bacterial cells that were injected into groundwater during a field experiment. The goal of the experiment was to investigate whether detachment of previously injected stained resident cells attached to aquifer sediment was enhanced in the presence of the newly injected mobile cells. This injection was an improvement on past experiments in that the attached (resident) cells were stained, allowing their concentrations to be enumerated directly by ferrographic capture (upon detachment). Contrary to expectations based on previous experiments, enhanced detachment of stained resident cells did not occur upon the arrival of injected cells. Consistent with previous experiments, however, was the observation of ephemeral increases in unstained cell concentrations coincident with the arrival of the stained injected cells. The ephemeral pulses of unstained cells were previously speculated to represent enhanced detachment of unstained indigenous cells in response to hydrodynamic collision with injected cells. The lack of enhanced detachment of stained resident cells in the present experiments indicates that increased concentrations of unstained cells may have occurred by mechanisms other than hydrodynamic collision. Visually observed variations in stain intensity indicated that increased unstained cell concentrations may have resulted from cell division at the low-concentration fringe of the injected plume.     

Measurement of fibroblast and bacterial detachment from biomaterials using jet impingement

Fibroblast and Staphylococcus aureus detachment strength from orthopaedic alloys and a tissue culture plastic (Thermanox) have been investigated with jet impingement. For S. aureus, unlike fibroblasts, detachment is caused more by pressure than shear. For these biomaterials, detachment strength is much higher for S. aureus than fibroblasts. Comparing materials under equivalent flow conditions, S. aureus attach to stainless steel and titanium with equal strength and more strongly than to Thermanox. For fibroblasts, detachment strength from all materials was similar. Fibroblast detachment strength from these biomaterials substantially decreases with time at equal flow rates and increases with flow rate at equal exposure times. Detachment strength is very similar for 3T3 and L929 fibroblasts on Thermanox for equivalent flow rate/time combinations, though enhanced adhesion of 3T3 cells was often noted for metals. Time effects are less evident for S. aureus. S. aureus adhesion to metals is more affected by flow rate than fibroblast adhesion.     

Dynamics of microbial communities on marine snow aggregates: colonization,growth, detachment,and grazing mortality of attached bacteria

We studied the dynamics of microbial communities attached to model aggregates (4-mm-diameter agar spheres) and the component processes of colonization, detachment, growth, and grazing mortality. Agar spheres incubated in raw seawater were rapidly colonized by bacteria, followed by flagellates and ciliates. Colonization can be described as a diffusion process, and encounter volume rates were estimated at about 0.01 and 0.1 cm(3) h(-1) for bacteria and flagellates, respectively. After initial colonization, the abundances of flagellates and ciliates remained approximately constant at 10(3) to 10(4) and approximately 10(2) cells sphere(-1), respectively, whereas bacterial populations increased at a declining rate to >10(7) cells sphere(-1). Attached microorganisms initially detached at high specific rates of approximately 10(-2) min(-1), but the bacteria gradually became irreversibly attached to the spheres. Bacterial growth (0 to 2 day(-1)) was density dependent and declined hyperbolically when cell density exceeded a threshold. Bacterivorous flagellates grazed on the sphere surface at an average saturated rate of 15 bacteria flagellate(-1) h(-1). At low bacterial densities, the flagellate surface clearance rate was approximately 5 x 10(-7) cm(2) min(-1), but it declined hyperbolically with increasing bacterial density. Using the experimentally estimated process rates and integrating the component processes in a simple model reproduces the main features of the observed microbial population dynamics. Differences between observed and predicted population dynamics suggest, however, that other factors, e.g., antagonistic interactions between bacteria, are of importance in shaping marine snow microbial communities.     

Experimental retinal detachment causes widespread and multilayered degeneration in rabbit retina

Retinal detachment remains one of the most frequent causes of visual impairment in humans, even after ophthalmoscopically successful retinal reattachment. This study was aimed at monitoring (ultra-) structural alterations of retinae of rabbits after experimental detachment. A surgical procedure was used to produce local retinal detachments in rabbit eyes similar to the typical lesions in human patients. At various periods after detachment, the detached retinal area as well as neighbouring attached regions were studied by light and electron microscopy. In addition to the well-known degeneration of photoreceptor cells in the detached retina, the following progressive alterations were observed, (i) in both the detached and the attached regions, an incomplete but severe loss of ganglion cell axons occurs; (ii) there is considerable ganglion cell death, particularly in the detached area; (iii) even in the attached retina distant from the detachment, small adherent groups of photoreceptor cells degenerate; (iv) these photoreceptor cells degenerate in an atypical sequence, with severely destructed somata and inner segments but well-maintained outer segments; and (v) the severe loss of retinal neurons is not accompanied by any significant loss of Müller (glial) cells. It is noteworthy that the described progressive (and probably irreparable) retinal destructions occur also in the attached retina, and may account for visual impairment in strikingly large areas of the visual field, even after retinal reattachment.     

Fluid shear contributions to bacteria cell detachment initiated by a monoclonal antibody

Receptor-mediated adhesion of bacteria to biological surfaces is a significant step leading to infection. Due to an increase in bacterial antibiotic resistance, novel methods to block and disrupt these specific interactions have gained considerable interest as possible therapeutic strategies. Recently, several monoclonal antibodies specific for the Staphylococcus aureus collagen receptor demonstrated specialized ability to displace attached cells from collagen in static assays. In this study, we experimentally examine the monoclonal antibody detachment functionality under physiological shear conditions to evaluate the role of this parameter in the detachment process. The detachment of staphylococci from collagen was quantified in real-time using a parallel plate flow chamber, phase contrast video-microscopy and digital image processing. The results demonstrate a unimodal dependence of detachment on fluid wall shear rate. The observed decrease in effective detachment rate with increasing force at the highest shear levels evaluated is counterintuitive and has not been previously demonstrated. Several possible mechanisms of this result are discussed.     

Bacterial adsorption to smooth surfaces: Rate, extent, and spatial pattern

The influence of bulk-water bacterial cell concentration and specific growth rate history on bacterial adsorption rates to surfaces was investigated using response surface analysis. A pure culture of Pseudomonas sp. 224S was grown in a chemostat and pumped into a continuous flow reactor where the bacteria were exposed to clean, glass surfaces under turbulent flow conditions for a period of six hours. Adsorption rate decreased approximately linearly with increasing specific growth rate history. Glass surfaces became saturated with 224S at ca. 0.1% coverage and the resulting spatial pattern of the adsorbed cells deviated from random in the direction of uniformity.     

The effects of cell density, attachment substratum and dexamethasone on spontaneous apoptosis of rat hepatocytes in primary culture

Summary The rates of spontaneous cell detachment, cell viability, and apoptosis in primary cultures of rat hepatocytes plated at high and low density were compared. Apoptosis was frequent in detached cells, and the rates of cell detachment and apoptosis were greater in high-density than in low-density cultures. Among attached cells, more cells had condensed or fragmented nuclei in high-density than in low-density cultures. Further, ladder-like DNA fragmentation was not seen in low-cell-density cultures but was clearly evident in high-density cultures. Bax was more highly expressed in cells cultured at high density, and on collagen vs. matrigel, whereas changes of Bcl-2 and Fas expression observed in culture appeared unrelated to the rate of apoptosis. The rate of hepatocyte apoptosis appeared to be identical in low-density cultures on collagen 1 and matrigel, but when cells were cultured at high density, matrigel suppressed apoptosis by more than 50% at 36 h. In hepatocytes cultured on collagen 1, dexamethasone (0.1 μM) suppressed apoptosis in both low- and high-density cultures; higher doses had no further effects. In high density cultures, aurintricarboxylic acid (10 μM) suppressed apoptosis and this improved cell attachment at 48 h. It is concluded that cell viability in primary cultures of rat hepatocytes grown on collagen I is dependent on optimal culture density and that the cell population is regulated, at least in part, by apoptosis. Corticosteroids suppress spontaneous apoptosis of cultured hepatocytes in a non-dose-dependent manner, whereas matrigel abolishes apoptosis induced by increasing cell density. Bax may be an important protein in the cell density and cell matrix-dependent regulation of apoptosis in cultured hepatocytes.     

Viscoelasticity of Staphylococcus aureus biofilms in response to fluid shear allows resistance to detachment and facilitates rolling migration

Staphylococcus aureus is a leading cause of catheter-related bloodstream infections and endocarditis. Both involve (i) biofilm formation, (ii) exposure to fluid shear, and (iii) high rates of dissemination. We found that viscoelasticity allowed S. aureus biofilms to resist detachment due to increased fluid shear by deformation, while remaining attached to a surface. Further, we report that S. aureus microcolonies moved downstream by rolling along the lumen walls of a glass flow cell, driven by the flow of the overlying fluid. The rolling appeared to be controlled by viscoelastic tethers. This tethered rolling may be important for the surface colonization of medical devices by nonmotile bacteria.     

Changes in cell-substratum adhesion and nuclear-cytoskeletal anchorage during cytodifferentiation of BeWo choriocarcinoma cells

Changes in the substratum anchorage of cells and nuclei were examined during methotrexate (MTX)-induced cytodifferentiation of BeWo human choriocarcinoma cells. During this process cytotrophoblast-like cells (CTLs) transform into giant mono- and multinucleated syncytiotrophoblast-like cells (STLs). Cells treated with MTX for 24 h exhibited significantly faster rates of substratum detachment by EDTA, trypsin-EDTA, EDTA-glycine, and DMSO than did uninduced controls. The decrease in cell-substratum adhesiveness occurred prior to the onset of morphological transformation. By 48 h, when morphological transformation was first observed, there had occurred a marked change in nuclear-cytoskeletal anchorage to the substratum, as evidenced by a difference in sensitivity of Triton-extracted STL and CTL monolayers to detachment by KI. STL monolayers were completely detached within 5 min of exposure to 0.3 M KI, while CTL monolayers remained firmly attached to the substratum for at least 3 h. KI-extracted residues were examined by electron microscopy and found to consist of nuclear shells attached to intermediate filaments. When cytoskeletal residues and KI-extracted proteins of STL and CTL cells were compared by two-dimensional polyacrylamide gel electrophoresis (PAGE), qualitative and quantitative differences were seen in a number of minor components. Thus the sensitivity of STL nuclear-cytoskeletal monolayers to removal by KI, an effective actin depolymerizing agent, may involve changes in the organization, stability, or interactions of actin with other components of the cytoskeletal framework.     

Influence of fluid shear and microbubbles on bacterial detachment from a surface

Prevention of microbial adhesion and detachment of adhering microorganisms from surfaces is important in many environmental, industrial, and medical applications. Fluid shear is an obvious parameter for stimulating microbial detachment from surfaces, but recently it has been pointed out that a passing air-liquid interface also has potential in stimulating microbial detachment. In the present study, the ability of microbubbles to stimulate detachment of bacterial strains from a glass surface is compared with the effects of fluid flow. Adhesion and detachment of Actinomyces naeslundii T14V-J1, Streptococcus oralis J22, and their coadhering aggregates were studied on glass, mounted in a parallel plate flow chamber. High fluid wall shear rates (11,000 to 16,000 s(-1)) were established in a laminar flow regime in the absence and presence of microbubbles. Wall shear rates stimulated detachment ranging from 70% to 30% for S. oralis and A. naeslundii, respectively. Coadhering aggregates were detached up to 54%. The presence of microbubbles in the flow increased the detachment of A. naeslundii within 2 min of flow from 40% in the absence of microbubbles to 98%, while detachment of neither S. oralis nor coadhering aggregates was affected by the presence of microbubbles. In summary, extremely high fluid flows can be effective in stimulating microbial detachment, depending on the strain involved. The addition of microbubbles to the flow allows the detachment of tenaciously adhering bacteria not detached by flow alone, but not of adhering coaggregates.     

Electron microscopic study of the methylcellulose-mediated detachment of cellulolytic rumen bacteria from cellulose fibers

The presence of methylcellulose prevents the attachment of cellulolytic rumen bacteria to cellulose fibers. The addition of methylcellulose to pure cultures of these organisms in which the cells are already adherent to cellulose causes their detachment from this insoluble substrate and the inhibition of their growth. Methylcellulose is not used as a carbon source by these organisms and has no effect on their growth when glucose and cellobiose are the carbon sources. Attached cells of Bacteroides succinogenes orient themselves in the plane of the individual cellulose fibers and their methylcellulose-induced detachment, which is complete (almost 100%), leaves grooves where the cellulose has been digested. Attached cells of Ruminococcus albus colonize the cellulose in a looser and less regular pattern and their almost complete methylcellulose-induced detachment leaves less regular pits in the cellulose surface. On the other hand, attached cells of Ruminococcus flavefaciens colonize the cellulose surface in a random orientation by means of a discernible exopolysaccharide network, and their less complete methylcellulose-induced detachment leaves no residual impressions on the cellulose surface. These data support the suggestion that bacterial attachment is necessary for the digestion of highly ordered crystalline cellulose, and that cellulolytic species differ in the nature of their attachment to this insoluble substrate and in the nature of their enzymatic attack. Methylcellulose is an effective agent for detaching major rumen cellulolytic bacteria from their cellulosic substrate.     

Oligomerized backbone pilin helps piliated Lactococcus lactis to withstand shear flow

The present work focuses on the role of pili present at the cell surface of Lactococcus lactis in bacterial adhesion to abiotic (hydrophobic polystyrene) and biotic (mucin-coated polystyrene) surfaces. Native pili-displaying strains and isogenic derivatives in which pilins or sortase C structural genes had been modified were used. Surface physico-chemistry, morphology and shear-flow-induced detachment of lactococcal cells were evaluated. The involvement of pili in L. lactis adhesion was clearly demonstrated, irrespective of the surface characteristics (hydrophobic/hydrophilic, presence or not of specific binding sites). The accessory pilin, PilC, and the backbone pilin, PilB, were revealed to play a major role in adhesion, provided that the PilB was present in its polymerized form. Within the population fraction that remained attached to the surface under increasing shear flow, different association behaviors were observed, showing that pili could serve as anchoring sites thus hampering the effect of shear flow on cell orientation and detachment.